Qualitative and quantitative screening of the ß-glucosidase activity in Saccharomyces cerevisiae and Saccharomyces uvarum strains isolated from refrigerated must.

The aim of the present work was to screen a pool of 75 yeasts belonging to the species Saccharomyces cerevisiae and Saccharomyces uvarum in order to select the strains endowed with ß-glucosidase activity. The first screening was a qualitative assay based on chromogenic substrates (arbutin and esculin). The second screening was the quantitative evaluation of the ß-glucosidase activity via a p-nitrophenyl-ß-d-glucopyranoside assay. The measurement was performed on three different cell preparations, including the extracellular compartment, the cell lysates and the whole cells. This study pointed out the high frequency of ß-glucosidase activity in S. uvarum strains. In particular, we retrieved three promising S. uvarum strains, CRY14, VA42 and GRAS14, featuring a high enzymatic activity, exploitable for winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: In yeasts, ß-glucosidase activity has been extensively described, especially in non-Saccharomyces species, while there is only little evidence of this activity in strains belonging to the Saccharomyces species. In winemaking, ß-glucosidase plays essential roles in the hydrolysis of glyco-conjugated precursors and the release of active aromatic compounds. This study provides new insights into the ß-glucosidase activity in strains belonging to Saccharomyces cerevisiae and Saccharomyces uvarum species, which are the most important strains in wine industry. Our results point out a marked enzymatic activity for the tested S. uvarum strains. These strains could be exploited for their potential ability to enhance the aroma profiles of wine. In addition, they could be potential sources for the commercial production of enzymes to be applied in winemaking.

Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs based on yeast breeding.

AIMS: The present work proposes a two-step molecular strategy to select inter- and intra-species Saccharomyces hybrids obtained by spore-to-spore mating, one of the most used methods for generating improved hybrids from homothallic wine yeasts. METHODS AND RESULTS: As low spore viability and haplo-selfing are the main causes of failed mating, at first, we used colony screening PCR (csPCR) of discriminative gene markers to select hybrids directly on dissection plate and discard homozygous diploid colonies arisen from one auto-diploidized progenitor. Then, pre-selected candidates were submitted to recursive streaking and conventional PCR in order to discriminate between the hybrids with stable genomic background and the false-positive admixtures of progenitor cells both undergone haplo-selfing. csPCRs of internal transcribed spacer (ITS) 1 or 2, and the subsequent digestion with diagnostic endonucleases HaeIII and RsaI, respectively, were efficient to select six new Saccharomyces cerevisiae × Saccharomyces uvarum hybrids from 64 crosses. Intragenic minisatellite regions in PIR3, HSP150, and DAN4 genes showed high inter-strain size variation detectable by cost-effective agarose gel electrophoresis and were successful to validate six new intra-species S. cerevisiae hybrids from 34 crosses. CONCLUSIONS: Both protocols reduce significantly the number of massive DNA extractions, prevent misinterpretations caused by one or both progenitors undergone haplo-selfing, and can be easily implemented in yeast labs without any specific instrumentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a method for the marker-assisted selection of several inter- and intra-species yeast hybrids in a cost-effective, rapid and reproducible manner.

A probabilistic method for computing quantitative risk indexes from medical injuries compensation claims.

BACKGROUND: The increasing demand of health care services and the complexity of health care delivery require Health Care Organizations (HCOs) to approach clinical risk management through proper methods and tools. An important aspect of risk management is to exploit the analysis of medical injuries compensation claims in order to reduce adverse events and, at the same time, to optimize the costs of health insurance policies. OBJECTIVES: This work provides a probabilistic method to estimate the risk level of a HCO by computing quantitative risk indexes from medical injury compensation claims. METHODS: Our method is based on the estimate of a loss probability distribution from compensation claims data through parametric and non-parametric modeling and Monte Carlo simulations. The loss distribution can be estimated both on the whole dataset and, thanks to the application of a Bayesian hierarchical model, on stratified data. The approach allows to quantitatively assessing the risk structure of the HCO by analyzing the loss distribution and deriving its expected value and percentiles. RESULTS: We applied the proposed method to 206 cases of injuries with compensation requests collected from 1999 to the first semester of 2007 by the HCO of Lodi, in the Northern part of Italy. We computed the risk indexes taking into account the different clinical departments and the different hospitals involved. CONCLUSIONS: The approach proved to be useful to understand the HCO risk structure in terms of frequency, severity, expected and unexpected loss related to adverse events.

Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway.

AIMS: An evolution-based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically, molybdate and chromate resistance was used as selectable phenotype to select sulfate permease-deficient variants that unable to produce sulfites and hydrogen sulfide (H(2) S). METHODS AND RESULTS: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the conjugation of gametes. Then, the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty-four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfite production (SO2 < 10 mg l(-1)) and with an impaired H2S production in grape must without added sulfites were selected. CONCLUSIONS: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly, resistance to toxic analogues of sulfate allowed us to detect strains that unable to assimilate sulfates. SIGNIFICANCE AND IMPACT OF THE STUDY: This strategy that combines the sexual recombination of spores and application of a specific selective pressure provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H2S.

Intrinsic gap and exciton condensation in the nu{T}=1 bilayer system.

We investigate the quasiparticle excitation of the bilayer quantum Hall (QH) system at a total filling factor nu{T}=1 in the limit of negligible interlayer tunneling under a tilted magnetic field. We show that the intrinsic quasiparticle excitation is of purely pseudospin origin and solely governed by the inter- and intralayer electron interactions. A model based on exciton formation successfully explains the quantitative behavior of the quasiparticle excitation gap, demonstrating the existence of a link between the excitonic QH state and the composite fermion liquid. Our results provide a new insight into the nature of the phase transition between the two states.

Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures.

AIMS: To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters. METHODS AND RESULTS: One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3.5, 3.2 and 3.0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery. CONCLUSIONS: The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.

Effects of extremely low-frequency magnetic fields on L-glutamic acid aqueous solutions at 20, 40, and 60 microT static magnetic fields.

Current peaks have been observed and measured in electrolytic ionic current of L-glutamic acid aqueous solutions at room temperature, in static magnetic fields of 20, 40, and 60 muT flux densities, with a superimposed extremely low-frequency, (1/10) Hz, alternating magnetic field flux density of 40 nT. The distributions of the peaks have mean values centered at the cyclotron resonance frequency of the singly charged L-glutamic acid ion molecular mass in the corresponding static field. Amplitudes and widths of the peaks are compared and analyzed to extract their correlation. The results can be considered a contribution to the understanding of the experimental phenomenology in low-frequency electromagnetic fields on ionic currents of L-glutamic amino acid aqueous solutions. The results can be of interest in the studies on the interaction of the electromagnetic fields with some structural neurotransmitters in cellular medium.

Effects of static and low-frequency alternating magnetic fields on the ionic electrolytic currents of glutamic acid aqueous solutions.

A current peak has been observed and measured in ionic electrolytic current of a glutamic acid aqueous solution, placed in a static magnetic field of flux density of 40 microT, with a superimposed low-frequency alternating magnetic field of flux density of 40 nT. The peak occurs at the frequency of the cyclotronic resonance of the molecular mass of a single charged glutamic acid ion, placed in a magnetic flux density equal to that of the static field. The amplitude of the current peak is about 30% of the background electrolytic current. Qualitative considerations and a listing of unsolved problems related to the phenomenology are given. The result is the first contribution to the study we have undertaken on the effects of low-frequency alternating electromagnetic fields on the ionic current of amino acid aqueous solutions which are the basic structural units of the proteins.

Spin-dependent phase diagram of the nuT=1 bilayer electron system.

We show that the spin degree of freedom plays a decisive role in the phase diagram of the nu(T)=1 bilayer electron system using an in-plane field B( parallel) in the regime of negligible tunneling. We observe that the phase boundary separating the quantum Hall and compressible states at d/l(B) = 1.90 for B(parallel) = 0 (d: interlayer distance, l(B): magnetic length) steadily shifts with B(parallel) before saturating at d/l(B) = 2.33 when the compressible state becomes fully polarized. Using a simple model for the energies of the competing phases, we can quantitatively describe our results. A new phase diagram as a function of d/l(B) and the Zeeman energy is established and its implications as to the nature of the phase transition are discussed.

Yeasts associated to Traditional Balsamic Vinegar: ecological and technological features.

Traditional Balsamic Vinegar (TBV) is an Italian homemade vinegar made with cooked grape must through a three-step process: conversion of sugars to ethanol by naturally occurring yeasts; oxidation of ethanol to acetic acid by acetic acid bacteria (AAB); and, finally, at least 12-years ageing. The cooked must is a selective and stressful medium for yeasts growth, due to its high sugar content and low pH values. Recent studies have shown that a large number of yeast species are involved in the fermentation, among them there are Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Hanseniaspora valbyensis, Hanseniaspora osmophila, Candida lactis-condensi, Candida stellata, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Nevertheless, the TBV-associated yeast population could be even more complex and many other slow-growing or poorly cultivable species might contribute to cooked must fermentation. In this review the main TBV yeast species are described, pointing out their role in TBV production and their influence on final product quality. Finally, both future developments in TBV yeast community studies (culture-independent and metagenomic techniques) and technological advances in TBV making (use of starter culture) are discussed.

Wine colour adsorption phenotype: an inheritable quantitative trait loci of yeasts.

AIMS: In this work, a population of 88 descendants derived from three wine strains of Saccharomyces cerevisiae was tested for the enological trait 'wine colour adsorption' (WCA) to evaluate its inheritability. METHODS AND RESULTS: The WCA phenotype was tested on plate agar medium specifically formulated for the purpose. After 10 days of anaerobic incubation at 28 degrees C, a computer-assisted assessment of WCA aptitude of the yeasts was carried out. The biomass colour -- ranging from white to dark brown -- reflects the adsorption of grape pigments: white and dark brown biomass colour corresponds to low and high adsorption, respectively. In order to confirm biomass colour results, microvinification trials using red must were performed, and the obtained wines were analysed. CONCLUSIONS: The analysis of the progeny demonstrated that the enological trait WCA is inheritable and polygenic. SIGNIFICANCE AND IMPACT OF THE STUDY: A way to describe the polygenic effect of the WCA trait has been found, also showing that this trait is inheritable. The impact of the work revolves more around the large-scale screening method, which could then assist in breeding wine yeast, and can also be used as a scientific tool to investigate WCA trait.

Molecular assessment of indigenous yeast population from traditional balsamic vinegar.

AIMS: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. METHODS AND RESULTS: Using the restriction analysis of the ribosomal region 5.8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5.8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. CONCLUSIONS: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.

Bacterial population in traditional sourdough evaluated by molecular methods.

AIMS: To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. METHODS AND RESULTS: Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. CONCLUSIONS: Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.

Occurrence and dominance of yeast species in sourdough.

AIMS: The aim of this work is to identify the dominant yeast species in homemade sourdoughs. METHODS AND RESULTS: PCR/restriction fragment length polymorphism analysis of internal transcribed spacer regions was used for the identification of isolates and the data were confirmed with phenotypic tests. The strains belonging to Saccharomyces cerevisiae were identified to strain level by analysis of inter-delta regions. CONCLUSION: This work shows that the dominant species in homemade sourdoughs can differ from each other. Saccharomyces cerevisiae was found to be the dominant species, followed by the Candida milleri, C. humilis, S. exiguus and Issatchenkia orientalis. The inter-delta regions of S. cerevisiae strains showed high polymorphism. SIGNIFICANCE AND IMPACT OF THE STUDY: Occurrence of single, non-Saccharomyces species and S. cerevisiae polymorphism in the yeast populations of sourdough samples.

Candida humilis--dominant species in sourdoughs for the production of durum wheat bran flour bread.

Yeasts present in the sourdough that is generally used for the production of durum wheat bran flour bread wereisolated and identified. Samples were taken during the rebuilding phase and at different intervals of time in order to monitor the population dynamics. The results obtained from the phenotypic studies were further confirmed by the molecular studies and enabled us to affirm that most of the strains, more than 95%, belong to the species Candida humilis. The dominance of C. humilis was steady in time. The isolations were carried out at sufficiently long intervals so that it was possible to ascertain that the conditions in which the sourdough is kept are fundamental to the microbiological stability of the dough.

Identification of Pichia anomala isolated from yoghurt by RFLP of the ITS region.

Several packs of swollen retailed plain and flavoured yoghurt were examined. The most commonly found species was Pichia anomala, identified both by physiological tests and RFLP analysis of the rDNA internal transcribed spacer (ITS) region. The isolated strains did not ferment lactose and were positive for galactose fermentation, confirming the hypothesis that galactose-fermenting yeast could be the cause of spoilage in yoghurt.

Saccharomyces uvarum, a proper species within Saccharomyces sensu stricto.

Saccharomyces uvarum is proposed as a proper species within the complex Saccharomyces sensu stricto. Molecular characteristics including the similarity of the restriction profile of the non-transcribed spacer 2 (NTS2) and of the D1/D2 sequences of the rDNA, as well as other genotypic and phenotypic characteristics confirm that this group of strains is highly homogeneous and distinguishable from other species of the Saccharomyces sensu stricto group.

Likelihood-ratio tests for hidden Markov models.

We consider hidden Markov models as a versatile class of models for weakly dependent random phenomena. The topic of the present paper is likelihood-ratio testing for hidden Markov models, and we show that, under appropriate conditions, the standard asymptotic theory of likelihood-ratio tests is valid. Such tests are crucial in the specification of multivariate Gaussian hidden Markov models, which we use to illustrate the applicability of our general results. Finally, the methodology is illustrated by means of a real data set.

Modelling categorical covariates in Bayesian disease mapping by partition structures.

We consider the problem of mapping the risk from a disease using a series of regional counts of observed and expected cases, and information on potential risk factors. To analyse this problem from a Bayesian viewpoint, we propose a methodology which extends a spatial partition model by including categorical covariate information. Such an extension allows detection of clusters in the residual variation, reflecting further, possibly unobserved, covariates. The methodology is implemented by means of reversible jump Markov chain Monte Carlo sampling. An application is presented in order to illustrate and compare our proposed extensions with a purely spatial partition model. Here we analyse a well-known data set on lip cancer incidence in Scotland.

Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto.

A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids.